In adipose cells, insulin induces the translocation of GLUT4 by stimulating their exocytosis from a basal intracellular compartment to the plasma membrane. Increasing overexpression of a hemagglutinin (HA) epitope-tagged GLUT4 in rat adipose cells results in a roughly proportional increase in cell-surface HA-GLUT4 levels in the basal state, accompanied by a marked reduction of fold HA-GLUT4 translocation in response to insulin. These results suggest that the mechanism involved in the intracellular sequestration of GLUT4 has a high capacity whereas the mechanism for GLUT4 translocation is readily saturated by overexpression of GLUT4, implicating an active translocation machinery in the exocytosis of GLUT4. We have analyzed the trafficking of HA-epitope-tagged GLUT4 mutants in transiently transfected primary rat adipose cells. Mutation of the C-terminal dileucine motif (LL489/90) does not affect the cell-surface expression of HA-GLUT4. However, mutation of the N-terminal phenylalanine-based targeting sequence (F5) results in substantial increases. Studies with wortmannin and coexpression of a dominant-negative dynamin GTPase mutant indicate that these effects appear to be primarily due to decreases and increases, respectively, in the rate of endocytosis. SNARE proteins are required for vesicle docking and fusion in eukaryotic cells in processes as diverse as homotypic membrane fusion and synaptic vesicle exocytosis [SNARE stands for SNAP receptor, where SNAP is soluble NSF attachment protein]. The SNARE proteins syntaxin 4 and vesicle-associated membrane protein (VAMP) 2/3 also participate in the insulin-stimulated translocaiton of GLUT4 from intracellular vesicles to the plasma membrane in adipose cells. We have now cloned and characterized rat SNAP-23, a ubiquitously expressed homologue of the essential neuronal SNARE protein SNAP-25 (synaptosomal-associated protein of 25 kDa). We demonstrate for the first time the participation of SNAP-23, along with syntaxin 4 and VAMP 2/3, in the formation of 20S Snare complexes prepared using rat adipose cell membranes and recombinant alpha-SNAP and NSF proteins. Agents that activate the G-protein G(i) (e.g. adenosine) increase, and agents that activate G(s) [e.g. isoprenaline (isoproterenol)] decrease, steady-state insulin-stimulated glucose transport activity and cell-surface GLUT4 in isolated rat adipose cells without changing plasma membrane GLUT4 content. Here we have further examined the effects of R(s)G(s) and R(i) G(i) ligands (in which R(s) and R(i) are G(s)- and G(i)- coupled receptors respectively) on insulin-stimulated cell-surface GLUT4 and the kinetics of GLUT4 trafficking in these same cells. The results suggest that R(s)G(s) and R(i)G(i) modulate insulin-stimulated glucose transport by influencing the extent to which GLUT4 is associated with occluded vesicles attached to the plasma membrane during exocytosis, perhaps by regulating the fusion process through which the GLUT4 in docked vesicles becomes exposed on the cell surface. We have also examined the effects of short-term exercise training on insulin-stimulated GLUT4 glucose transporter translocation and glucose transport activity in rat adipose cells. The results suggest that increases in total GLUT4 and GLUT4 translocation to the cell surface contribute to the increase in maximally insulin-stimulated glucose transport with short-term exercise training. In addition, the results suggest that the exercise training-induced adaptations in glucose transport occur more rapidly than previously thought and with minimal changes in adipose cell size.